Pcdna 1 myc his empty vector

NIH/3T3 and C2C12 cells (1.5 × 10⁴) were seeded for luciferase reporter gene assay in a 96-well plate. BRE₂-Luc or the CAGA₁₂-Luc plasmids were transfected together with pcDNA.1/myc-His(−) empty vector (Thermo Fisher Scientific), mIgsf1-myc-His or isoform 2 ARHGAP36-N-mCherry^{25 (link)} using Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer’s instructions. A constitutively expressing construct encoding renilla luciferase (RL-TK, Promega) was co-transfected as internal control. The next day, cells were starved in serum-free DMEM for four hours and stimulated with BMP2 or TGFβ1 overnight. Cell lysis was performed using passive lysis buffer (Promega) and measurement of luciferase activity was carried out according to the manufacturer’s instructions using a TECAN infinite f200 Luminometer. Data are shown as relative light units (RLU) normalized to the empty vector control. The experiments were performed with n = 3, 4, or 5 technical replicates, as stated in respective figure legends.

NIH/3T3 and C2C12 cells (1.5 × 10⁴) were seeded for luciferase reporter gene assay in a 96-well plate. BRE₂-Luc or the CAGA₁₂-Luc plasmids were transfected together with pcDNA.1/myc-His(−) empty vector (Thermo Fisher Scientific), mIgsf1-myc-His or isoform 2 ARHGAP36-N-mCherry^{25 (link)} using Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer’s instructions. A constitutively expressing construct encoding renilla luciferase (RL-TK, Promega) was co-transfected as internal control. The next day, cells were starved in serum-free DMEM for four hours and stimulated with BMP2 or TGFβ1 overnight. Cell lysis was performed using passive lysis buffer (Promega) and measurement of luciferase activity was carried out according to the manufacturer’s instructions using a TECAN infinite f200 Luminometer. Data are shown as relative light units (RLU) normalized to the empty vector control. The experiments were performed with n = 3, 4, or 5 technical replicates, as stated in respective figure legends.

NIH/3T3 and C2C12 cells (1.5 × 10⁴) were seeded for luciferase reporter gene assay in a 96-well plate. BRE₂-Luc or the CAGA₁₂-Luc plasmids were transfected together with pcDNA.1/myc-His(−) empty vector (Thermo Fisher Scientific), mIgsf1-myc-His or isoform 2 ARHGAP36-N-mCherry^{25 (link)} using Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer’s instructions. A constitutively expressing construct encoding renilla luciferase (RL-TK, Promega) was co-transfected as internal control. The next day, cells were starved in serum-free DMEM for four hours and stimulated with BMP2 or TGFβ1 overnight. Cell lysis was performed using passive lysis buffer (Promega) and measurement of luciferase activity was carried out according to the manufacturer’s instructions using a TECAN infinite f200 Luminometer. Data are shown as relative light units (RLU) normalized to the empty vector control. The experiments were performed with n = 3, 4, or 5 technical replicates, as stated in respective figure legends.